The development of rugged, easy-to-use fluorescent methods for analyte detection is essential for environmental monitoring, high-throughput screening of drug candidates, detection of microorganisms in the environment and disease diagnosis. The increased reliance on these assays necessitates the development of more stable, sensitive and robust detection methods to reduce the time-consuming and expensive impact of false positives and negatives. This project will develop a new fluorescent assay using a variation of the traditional indicator displacement assay (IDA) for analyte detection by using a biological host for indicator-guest recognition. The use of a biological host has two distinct advantages: (1) synthetic hosts, such as cyclodextrans, metal complexes, cucurbiturils and calixarenes, lack the selectivity, specificity, binding kinetics, and nanomolar binding constants of biological systems, and (2) the larger molecular weight and hydrophobic interior of a biological host would allow us to exploit additional signaling methodologies, such as fluorescence anisotropy, lifetime and ratiometric methods. We will (1) develop a novel assay based on the ability to manipulate a fluorescent signal by using a non-fluorescent reactive guest to block coordination of an environmentally sensitive indicator to the biological host (2) demonstration of assay versatility by creation of new reactive guest molecules and (3) incorporation of our assay into high throughput screening technologies.